Platelet activation is facilitated by the mobilization of arachidonic acid from endogenous phospholipid storage depots and the subsequent generation of biologically active eicosanoid metabolites. During the current grant interval, we have cloned and expressed the major platelet phospholipase A2 and have demonstrated that catalysis proceeds through the formation of a novel arachidonoyl-enzyme intermediate. Although traditional dogma has emphasized the role of random diffusion of released arachidonic acid in the metabolism and biologic action of eicosanoids, the identification of the arachidonoyl-phospholipase A2 intermediate suggests: 1) the temporally and spatially orchestrated generation and shuttling of eicosanoids in activated cells; 2) the transacylation of arachidonic acid to a variety of nucleophilic acceptors during cellular stimulation; and 3) a novel mechanism for incorporation of exogenous nonesterified arachidonic acid into cellular lipids. Accordingly, the overall goal of the first two sections of proposed research is the identification of the biochemical mechanisms which: 1) regulate the formation and hydrolysis of the arachidonoyl-enzyme intermediate; 2) direct the metabolic fate of the covalently thioesterified arachidonate; and 3) mediate the trafficking of arachidonate to downstream proteins for either oxidation or transport. These goals will be accomplished through utilizing recombinant purified phospholipases (e.g., 30kDa or 85kDa polypeptides), recombinant cyclooxygenase and other essential factors as necessary to characterize and reconstitute arachidonic acid release and eicosanoid production in purified systems comprised of the minimal number of homogeneous constituents. Finally, our recent advances in electrospray mass spectrometry of phospholipids will be exploited to study lipid turnover in activated platelets with unparalleled specificity, resolution and mechanistic insight. Collectively, these studies will provide important new information on the diversity, regulation and function of a novel family of proteins mediating the orchestration of arachidonic acid mobilization during cellular activation which will have profound effects on our understanding and treatment of thrombosis, atherosclerosis and inflammation.